ABSRACT Coronary heart disease is the leading cause of morbidity and mortality worldwide. Timely restoration of coronary perfusion known as reperfusion is the only effective therapeutic intervention for protecting the heart from myocardial infarction (MI). Currently, there is no effective therapy for preventing cardiac ischemia-reperfusion (IR) injury. The loss of mitochondrial function plays a crucial role in IR-induced cell death suggesting that protection and restoration of mitochondrial function is pivotal to cell survival in the heart. However, limited knowledge of the mechanisms underlying mitochondria-mediated cell death obscures the development of new mitochondria-targeted cardioprotective compounds. Cardiac IR increases calcium, reactive oxygen species (ROS), and inorganic phosphate levels in mitochondria that induce mitochondrial permeability transition (MPT) concurrently with opening of the non-specific pathological MPT pores in the inner mitochondrial membrane. High mitochondrial ROS (mtROS) also may disintegrate mitochondrial supercomplexes (SCs), predominantly due to oxidation of cardiolipin (CL), a unique mitochondrial phospholipid. SCs are large supramolecular complexes containing individual electron transport chain (ETC) complexes. According to the solid-state model, the assembly of SCs provides high-efficiency electron flux throughout the ETC; it increases ATP synthesis and significantly reduces electron leakage and mtROS production due to short diffusion distances between ETC complexes. The cause-and-effect relationship between MPT induction and SC degradation has not yet been established. We hypothesize that the MPT plays a causal role in the disintegration of mitochondrial SCs, leading to diminished energy metabolism and cell death in cardiac IR. We propose that MPT-induced mitochondrial swelling sensitizes CL to the ROS attack leading to degradation of SCs. The Specific Aims are as follows: (1) Determine the timing of MPT, disintegration of SCs and progression of post-MI injury. We will investigate the association between MPT pore opening and SC degradation, with progression of IR injury, using the animal model of in vivo MI induced by coronary artery ligation with/without subsequent reperfusion. We will also subject WT and tafazzin knockdown (TazKD) mice to cardiac IR to distinguish changes in SC assembling induced by CL oxidation versus CL deficiency. (2) Examine the cause-and-effect relationship between the MPT induction and SC disintegration in response to oxidative stress. We will subject cardiomyocytes/mitochondria with CyP-D (Ppif) and/or tafazzin deficiency to oxidative stress to clarify a cause-and-effect relationship between MPT and SC disintegration. In addition, cardiac mitochondria will be treated with oxidized CL in the presence of MPT inducers/blockers to reveal a causal role of MPT versus CL oxidation in SC degradation. (3) Define if inhibition of MPT, and mtROS scavenging protect synergistically against post-MI injury. These studies will establish whether combined therapy simultaneously targeting mtROS and MPT exerts synergistic cardioprotective effects on cardiac IR. The MPT inhibitor will be administered alone or in combination with mtROS scavengers during in vivo cardiac IR. In addition, WT and TazKD hearts will be subjected to ex vivo IR in the presence of the MPT inhibitor and/or mtROS scavengers. Overall, elucidating the crosstalk mechanisms between MPT and SC degradation will provide new insights into the molecular basis of mitochondria-mediated cell death during cardiac IR. The outcome of this project will allow development of new therapeutic strategies to prevent myocardial IR injury, and improve clinical consequences in patients with acute myocardial infarction through targeting mitochondria.